Enhanced protein phosphorylation is one of the intracellular events which invariably follow membrane receptor activation. Recently identified protein kinase C (also termed calcium- and phospholipid-dependent protein kinase) is a unique enzyme that phosphorylates specific proteins whose functions remain to be determined. Protein kinase C (PKC) requires a phospholipid and a diacylglycerol (DAG) for maximal activity. DAG is the hydrolytic product of membrane polyphosphoinositide breakdown, which occurs following membrane receptor occupancy, and is one of the initial events in signal transduction. The release of DAG during PI turnover substantially stimulates PKC activity over that observed in quiescent cells. The known co-carcinogen or tumor promotor TPA specifically increases membrane PKC activity. Associated with this increased membrane PKC activity is a reduction of cytosolic PKC activity, which suggests that the translocation of PKC is an important intracellular event. Using a homogeneous population of cells (a mouse-derived, anterior pituitary tumor cell line [AtT-20s]) that respond to a variety of membrane and intracellular activators by releasing POMC-derived peptides, we have explored the involvement of PKC in hormone secretion by monitoring the release of beta-endorphin (BE) as well as membrane and cytosolic PKC activity. TPA treatment resulted in a dose-related increase in BE secretion and translocation of PKC from the cytosolic to the membrane fraction. A maximally stimulating concentration of TPA enhanced membrane PKC activity twofold within 1 minute and fivefold within 3 minutes. It would appear that activation (translocation) of PKC results in enhanced hormone release through a pathway that does not involve the adenylate cyclase/cAMP system.